The BCL2 family proteins function in cell life and death decisions by regulating and affecting the process of mitochondrial outer membrane permeabilization (MOMP) in apoptosis. This protein family also regulates other processes, independently of MOMP, such as mitochondrial dynamics, calcium homeostasis, autophagy, and metabolism. We have defined different modes of canonical and non-canonical BCL2 family-mediated cell survival. These include sequestration of direct activators of the BCL2 effectors, BAX and BAK (Mode 1) and of the activated effectors themselves (Mode 2). We have also characterized a non-canonical effector, BOK, which is not controlled by anti-apoptotic BCL2 family proteins but rather by the ERAD pathway, which induces its degradation. In this renewal application (representing our only support for studies on the BCL2 family), we hypothesize that the ways in which the BCL2 proteins prevent MOMP and apoptosis impacts on cellular physiology via the other, MOMP-independent effects of these proteins. This, in turn, influences the process of MOMP and apoptosis when the survival mechanisms are perturbed. Based on our published and preliminary results, and using novel approaches to interrogate events at the single cell level, we will ask: 1. How do different modes of canonical BCL2 family-mediated survival impact cellular function independently of MOMP? Here we will explore how different modes of BCL2 family survival states in living cells affect cellular processes independently of their direct effects in controlling MOMP. In particular, we will examine mitochondrial dynamics, autophagy, and calcium homeostasis at the endoplasmic reticulum. 2. How do different modes of canonical BCL2 family interactions integrate cell homeostasis and stress signals to promote cellular survival or cell death? Here we will explore the roles and effects of different survival states controlled by BCL2 family proteins and how their perturbation leads to MOMP and cell death. In this way we will determine how the physiological effects of BCL2 protein interactions influences cell death and survival upon de-repression of the anti-apoptotic BCL2 proteins. We will also examine, at the single cell level, how directly manipulating BCL2 family interactions in living cells impacts on signaling states in cells that persist following stress. 3. How does a noncanonical BCL2 family survival state impact cellular physiology and cell death? Here we will investigate the functions of the BCL2 effector, BOK. We have found that BOK is capable of directly causing MOMP, but this apoptotic activity is not effectively inhibited by anti-apoptotic BCL2 proteins. Instead, it is inhibited by the cytosolic arm of the ER-associated degradation (ERAD) pathway. The effects of both unstable and stabilized BOK on apoptosis, calcium homeostasis, and metabolism will be explored. Our program will form the basis for a new, integrated understanding of how the BCL2 family functions in cell physiology.